The long-term goal of this investigation is to identify genetic predispositions to complications of alcoholism. An abnormality of the thiamin pyrophosphate (TPP)-dependent enzyme transketolase (EC 2.2.1.1) has been identified in patients with Wernicke-Korsakoff syndrome, a disease found primarily in a subgroup of alcohol abusers who respond favorably on thiamin treatment. In the past one and a half years we have identified a functionally impaired transketolase protein in cultured skin fibroblasts from three patients with Wernicke-Korsakoff syndrome, by enzymatic and immunochemical techniques. Molecular cloning for the transketolase cDNA is well underway. It has been proposed that the abnormality of transketolase is caused by an alteration of the transketolase gene. We plan to elucidate the molecular basis of the transketolase abnormality in these three fibroblast lines that the protein is already shown to be abnormal, an to other patients and non-symptomatic family members. The results may lead to a better understanding of the genetic predisposition, as well as the interaction of gene and nutrition (thiamin) in the development of Wernicke-Korsakoff syndrome. We plan to further describe the abnormality of transketolase enzymatically and immunochemically at the protein level in these three Wernicke-Korsakoff cells, and extend these biochemical work to other fibroblasts from Wernicke-Korsakoff patients and non-symptomatic alcoholic controls. The protein sequence will be obtained by conventional molecular biological techniques. Complementary DNA will be isolated and cloned from the human fibroblast cDNA expression library using the antibody probe, and verified by matching for partial amino acid sequence we already obtained. The transketolase cDNA in the Wernicke-Korsakoff and non-symptomatic alcoholic control fibroblasts will be amplified by polymerase chain reaction (PCR) method, using the mRNA/cDNA hybrid prepared form these cells and well-spaced oligonucleotide primers. The transketolase cDNA from patient and control cells will be compared by direct sequencing of the PCR-amplified, total heterozygous cDNA, and each of the allelic, clonal cDNA. The abnormality of transketolase in these Wernicke-Korsakoff fibroblasts will be interpreted based on the genetic alteration of protein sequence.